Field of the Invention
The present invention relates to identification of unique identifying sequences and taxa which can identify specific organism sources of contamination in samples, especially environmental samples, and methods and compositions that find use thereof.
Related Art
Beach closures and public health advisories have a major economic impact on coastal communities whose economies are based largely on tourism from beach recreation. Most closings and advisories are triggered by water samples that exceed microbial water quality standards for fecal indicator bacteria (FIB), usually culturable coliforms, E. coli or enterococci that are considered a proxy for human health risk in recreational waters. Because the direct measurement of all human pathogens is often impractical and unreliable under field conditions, water monitoring relies on the detection of bacterial indicators that have some demonstrated correlation with human illness in areas mostly impacted by human sewage (Field, K. G.; Samadpour, M., Fecal source tracking, the indicator paradigm, and managing water quality. Water Research 2007, 41, (16), 3517-3538; Wade, T. J.; Pai, N.; Eisenberg, J. N. S.; Colford, J. M., Do US Environmental Protection Agency water quality guidelines for recreational waters prevent gastrointestinal illness? A systematic review and meta-analysis. Environmental Health Perspectives 2003, 111, (8), 1102-1109). Sewage, however, is one of many potential sources of FIB, and monitoring results are often confounded by inputs from a variety of wildlife and non-fecal sources (Field, K. et al., Water Research 2007, 41, (16), 3517-3538; Boehm, A. B., Enterococci concentrations in diverse coastal environments exhibit extreme variability. Environmental Science & Technology 2007, 41, (24), 8227-8232; Boehm, A. B., Covariation and Photoinactivation of Traditional and Novel Indicator Organisms and Human Viruses at a Sewage-Impacted Marine Beach. Environmental Science & Technology 2009, 43, (21), 8046-8052; Yamahara, K. M.; Layton, B. A.; Santoro, A. E.; Boehm, A. B., Beach sands along the California coast are diffuse sources of fecal bacteria to coastal waters. Environmental Science & Technology 2007, 41, (13), 4515-4521). FIB are common in most warm-blooded animals, and many studies demonstrate that FIB occur in several environmental sources aside from feces, including soils and sediments, algal wrack and beach sands. Ibid. Thus water bodies often contain measurable amounts of FIB even where anthropogenic inputs are absent, and the presence of FIB provides an insufficient indication of health risk without additional source tracking data.
Shortcomings of the current FIB monitoring approach combined with widespread development and implementation of Total Maximum Daily Load (TMDL) requirements for microbiological pollution are fueling interest in microbial source tracking (MST) methods (Santo Domingo, J. W.; Bambic, D. G.; Edge, T. A.; Wuertz, S., Quo vadis source tracking? Towards a strategic framework for environmental monitoring of fecal pollution. Water Research 2007, 41, (16), 3539-52; USEPA, Microbial Source Tracking Guide Document. In Washington, D.C., 2005; p 131). Many approaches to source tracking are under development, most of which rely on single phenotypic or genotypic biomarkers to measure sources (Field, K., et al., Water Research 2007, 41, (16), 3517-3538; Santo Domingo, J. W. et al., Quo vadis source tracking? Towards a strategic framework for environmental monitoring of fecal pollution. Water Research 2007, 41, (16), 3539-52). A limitation of single targets is that no single assay is known to be 100% specific for any one type of waste (Field, K., et al., Water Research 2007, 41, (16), 3517-3538; Santo Domingo, J. W. et al., Quo vadis source tracking? Towards a strategic framework for environmental monitoring of fecal pollution. Water Research 2007, 41, (16), 3539-52), and MST based on single targets is entirely dependent on the fate of one biomarker once it enters receiving waters (Bae, S.; Wuertz, S., Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide. Water Research 2009, 43, (19), 4850-4859; Balleste, E.; Blanch, A. R., Persistence of Bacteroides Species Populations in a River as Measured by Molecular and Culture Techniques. Applied and Environmental Microbiology 2010, 76, (22), 7608-7616; Walters, S. P.; Field, K. G., Survival and persistence of human and ruminant-specific fecal Bacteroidales in freshwater microcosms. Environmental Microbiology 2009, 11, (6), 1410-1421).
A huge diversity of microorganisms is resident in human and animal guts. Approximately 1000 different microbial taxa are now known to reside in the human gut alone, but the potential for this diversity to be used as a means for identifying sources remains largely unexplored and there have been few comparative surveys of microbial community composition among important sources of fecal contamination (Cao, Y.; Wu, C. H.; Andersen, G. L.; Holden, P. A., Community analysis-based methods. In Microbial Source Tracking: Methods, Applications, and Case Studies, Hagedorn, C.; Blanch, A. R.; Harwood, V. J., Eds. Springer: New York, N.Y., 2011; pp 251-282; Lee, J. E.; Lee, S.; Sung, J.; Ko, G., Analysis of human and animal fecal microbiota for microbial source tracking. The ISME journal 2011, 5, (2), 362-5; Unno, T.; Jang, J.; Han, D.; Kim, J. H.; Sadowsky, M. J.; Kim, O. S.; Chun, J.; Hur, H. G., Use of Barcoded Pyrosequencing and Shared OTUs To Determine Sources of Fecal Bacteria in Watersheds. Environmental Science & Technology 2010, 44, (20), 7777-7782). New techniques for high-throughput DNA sequence analysis such as high-density microarrays and next-generation sequencing (NGS) technologies like pyrosequencing are enabling comprehensive surveys of diverse microbial communities that occur in a sample. Targeting the whole microbial community for source identification is a fundamentally different approach than traditional molecular methods that are dependent on the detection on one gene sequence under complex environmental conditions (Cao, Y.; Wu, C. H.; Andersen, G. L.; Holden, P. A., Community analysis-based methods. In Microbial Source Tracking: Methods, Applications, and Case Studies, Hagedorn, C.; Blanch, A. R.; Harwood, V. J., Eds. Springer: New York, N.Y., 2011; pp 251-282; Wu, C. H.; Sercu, B.; Van de Werfhorst, L. C.; Wong, J.; DeSantis, T. Z.; Brodie, E. L.; Hazen, T. C.; Holden, P. A.; Andersen, G. L., Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators. PLoS One 2010, 5, (6), e11285; Cao, Y. P.; Van De Werfhorst, L. C.; Sercu, B.; Murray, J. L. S.; Holden, P. A., Application of an Integrated Community Analysis Approach for Microbial Source Tracking in a Coastal Creek. Environmental Science & Technology 2011, 45, (17), 7195-7201; Jeong, J. Y.; Park, H. D.; Lee, K. H.; Weon, H. Y.; Ka, J. O., Microbial Community Analysis and Identification of Alternative Host-Specific Fecal Indicators in Fecal and River Water Samples Using Pyrosequencing. Journal of Microbiology 2011, 49, (4), 585-594). Sequence analysis of entire microbial communities creates an opportunity to discover a multitude to different bacterial species that are unique to fecal and environmental sources of FIB in recreational waters.